Vesicle-enriched secretomes alter bacterial competitive abilities and are drivers of evolution in microbial communities

Abstract Microbial membrane vesicles can carry compounds that inhibit bacterial growth, but how they impact the fitness of the vesicle-producing bacterial species and influence community dynamics remain unexplored questions. To address these questions, we examined the effect of vesicle-enriched secretomes (VESs) in different single-species and multi-species systems. Effects of VESs on single-species growth dynamics were determined for nine bacterial species belonging to four genera (Escherichia, Salmonella, Pseudomonas and Bacillus) in nutrient-rich and poor growth media. Results showed both species-specific and nutrient-dependent effects of the VESs on bacterial growth. The strongest antagonistic effects were observed for VES isolated from the natural isolates of E. coli, while those isolated from P. aeruginosa PA14 affected the highest number of species. We further demonstrated that these VESs altered the competitive abilities of the species involved in two-species (S. Typhimurium LT2 and S. arizonae) and three-species systems (E. coli, S. Typhimurium LT2 and B. subtilis). Finally, using experimental evolution we showed that different bacterial species could rapidly acquire mutations that abrogated the antagonistic effects of VESs. This study demonstrates how VESs can contribute in shaping microbial communities, both by increasing the competitive ability of a given bacterial species and as a driver of genetic adaptation.


Introduction
One of the most common ways for bacteria to interact with one another is by secr eting differ ent molecules (e .g. nutrients , enzymes , pheromones , or antibacterial agents) in the surrounding environment (Lamont et al. 2002, Nützmann et al. 2011, Traxler et al. 2013 ).The most well-studied mechanisms employed by bacteria to secrete these molecules in the environment include specific secretion systems (Bleves et al. 2010, Hood et al. 2010, Cianfanelli et al. 2016, Wexler et al. 2016 ), or in some cases cell l ysis (Cav ard 2004, Mader et al. 2015 ).In this study, we determined the role of the secretory system comprised of membrane vesicles in microbial interactions (Fig. 1 ).Membrane vesicles are spherical bilipid layered structures, 20-250 nm in size, that are released by all bacteria by blebbing out of the membrane and that can secrete different types of molecules (Schwechheimer and Kuehn 2015 , P athir ana and Ka par akis-Liask os 2016 , Reidl 2016, Roier et al. 2016, Jan 2017 ).These v esicles typicall y carry different types of molecules either within, embedded in the membrane, or bound on the outer surface of the vesicles (Dorw ar d and Garon 1990, Horstman and Kuehn 2000, Roier et al. 2016, Johnston et al. 2023 ).
Studies that have investigated the association of these vesicles to different bacterial phenotypes have either used purified vesicles (Dorw ar d and Garon 1990, Horstman and Kuehn 2000, Biller et al. 2014, Koeppen et al. 2016 ) or secretomes that are enriched for vesicles (Kadurugamuwa and Beveridge 1995, 1996, Evans et al. 2012, K oeppen et al. 2016, Zakharzhevska ya et al. 2017, Nice et al. 2018, Johnston et al. 2023 ) (fr om now on r eferr ed to as v esicleenric hed secr etomes or VESs, see Materials and Methods ), with results from both these approaches offering novel insights about the biological significance of these vesicles .T hus , several studies have shown membrane vesicles and vesicle-enriched secretomes to be important in host-pathogen interactions, and in defense against antibiotics and phages (Kadurugamuwa and Beveridge 1995 , Kesty et al. 2004 , Ka par akis-Liask os and Ferr er o 2015 , Koeppen et al. 2016, Olsen et al. 2016, Zakharzhevskaya et al. 2017, Nice et al. 2018, Reyes-Robles et al. 2018, Balhuizen et al. 2021, Dhital et al. 2022 ).Although these studies have increased our understanding of the regulation, biogenesis, and medical rele v ance of the vesicles and v esicle-enric hed secr etomes, if and how these affect a bacterial's competitive ability and shape micr obial comm unities r emain unclear.A fe w studies inv estigating the role of vesicles and vesicle-enriched secretomes in microbemicr obe inter actions hav e demonstr ated both anta gonistic and synergistic effects associated with these vesicles.Important examples of the former are vesicle-enriched secretomes isolated from Pseudomonas aeruginosa that were shown to be antagonistic to w ar ds both Gr am-negativ e and Gr am-positiv e bacterial species (Kadurugam uwa and Be v eridge 1996 ), and those isolated from the predatory bacterium Myxococcus xanthus that carry hydr ol ytic en- zymes with antibacterial activity (Evans et al. 2012 ).An example of the latter are vesicles isolated from the marine bacterium Prochlorococcus that facilitate the growth of other marine bacteria (Alteromonas and Halomonas) by acting as a carrier of carbon compounds across the marine microbial community (Biller et al. 2014 ).Furthermor e, membr ane v esicles can also induce physiological changes in the surrounding bacteria as was demonstrated in the interactions between Corynebacterium durum and Streptococcus sanguinis , where the fatty acid containing vesicles from the former induced cell-chain morphology in the latter (Treerat et al. 2020 ).Vesicles may also aid in extracellular heme cycling as was demonstrated for the Gram-positive bacterium Dietzia sp.DQ12-45-1b (Wang et al. 2021 ).Although these studies demonstrate an association of vesicles to various microbial phenotypes, most of them hav e onl y inv estigated the effects of these v esicles in isolation or in single-species systems.
To examine the r ele v ance of v esicle-enric hed secr etomes in two-and thr ee-species comm unities , we in vestigated how the fitness of a bacterial species changed with different levels of its VESs, and how bacteria ada pted and e volv ed in r esponse to these c hanges.Suc h fitness-phenotypes ma ps hav e been widel y used in evolutionary biology to determine the potential for a trait to e volv e under selection and to understand the tempo and mode of ada ptiv e e volution (Dykhuizen et al. 1984, Hartl et al. 1985, Lunzer et al. 2005, Dalziel et al. 2009, Natarajan et al. 2018, Velotta et al. 2018 ), and are instrumental in highlighting the potential for these vesicles to affect interactions in microbial communities.Our results show that the antagonistic effects of these VESs, depending on bacterial species, could shape the community composition and that bacteria can r a pidl y e volv e to r esist these anta gonistic effects .T hese findings suggest that VESs r epr esent an important and unexplored form of selective pressure that can influence micr obial comm unities.

Bacterial strains and culture conditions
Nine different bacterial species were used in this study, including E. coli K-12 MG1655, ECOR 5, ECOR 11 and ECOR 36 (the last three ar e natur al isolates of E. coli fr om the ECOR collection), Salmonella enterica subsp.typhimurium LT2 (henceforth called S. Typhimurium LT2), Salmonella enterica subsp.arizonae (henceforth called S. arizonae ), Pseudomonas aeruginosa PA01, Pseudomonas aeruginosa PA14 and Bacillus subtilis .The nutrient-rich media used in these experiments was Lysogeny broth (LB) and the nutrient-poor me-dia was M9-minimal media containing 0.2% glucose (Leach 1992 ).All strains were grown at 37 • C. For long-term storage of strains, ov ernight-gr own cultur es wer e mixed with DMSO (9 : 1) and were stored at −80 • C.

Isolation of v esicle-enric hed secretome (VES)
Vesicle-enric hed secr etomes wer e isolated by ada pting the methods described pr e viousl y (McBr oom andKuehn 2007 , McMillan andKuehn 2022 ).Briefly, each bacterial species was grown in 50 ml of a ppr opriate medium for 16 hrs (bacterial cultur e densities after 16 hrs of growth is mentioned in T able S1).T o obtain a cell-free supernatant 40 ml of the culture was then centrifuged at 4500 r/m for 30 mins .T he supernatant was then filtered through a 0.45 μM syringe filter and was then centrifuged again at 40,000 g for 3 hrs.After 3 hrs the supernatant was car efull y discarded and the pelleted v esicle-enric hed secr etomes wer e washed with phosphatebuffered saline (PBS) once, follo w ed b y another round of centrifugation at 40,000 g for 3 hrs .T he final pellet was dissolved in 5 ml of the a ppr opriate medium and then filter sterilized again using a 0.45 μM syringe filter.The sterility of the final vesicle preparation was c hec ked by spotting 100 ul on LA plates .T he concentrations and size of the vesicles were checked by performing nanoparticle tr ac king anal ysis (NTA) (Table S2).NTA was performed on the Nanosight LM10 instrument, which uses a laser beam to illuminate the surface where nanoparticle-sized particles in the suspension can be counted (Malloy and Carr 2006 ).Each VES was diluted 1 : 100 in phosphate-buffered saline, and the measurement was performed in two 30-seconds frames with the average of these measur ements r ecor ded.Tw o biological replicates w ere used in each case.
Although different studies have used different speeds of centrifugation to isolate vesicles, we used 40,000 g for 3 hrs, as this has been shown to be sufficient for the isolation of membrane v esicles (McBr oom andKuehn 2007 , McMillan andKuehn 2022 ).The presence of vesicles in these preparations is confirmed by performing cryo-EM investigation of VESs from ECOR 36 ( Fig. S5 ).Specimen for cryo-EM investigation was pr epar ed at 25 • C and high humidity within a custom-built environmental chamber.A small ( ∼1 μL) drop of the sample was deposited on a carbon-sputtered copper grid (300 mesh, Agar scientific) cov er ed with a perforated polymer film.Excess liquid was thereafter removed by blotting with a filter paper.This leaves a thin film of the solution on the grid.The sample was immediately vitrified in liquid ethane and tr ansferr ed to the micr oscope, continuousl y kept below −160 • C, and pr otected a gainst atmospheric conditions.Anal yses wer e performed with a Zeiss Libra 120 transmission electron microscope (Carl Zeiss A G , Oberk oc hen, German y) oper ating at 80 kV and in zero-loss bright-field mode.Digital images were recorded under low-dose conditions with a BioVision Pro-SM Slow Scan CCD camer a (Pr oscan Elektr onisc he Systeme GmbH, Sc heuring, Germany).
The aim of our study was to determine the effects of both the isolated vesicles, as well as of molecules that are enriched with these vesicles, on bacterial interactions .T hus , we only wash the vesicles with PBS to remove any loosely bound components, befor e r e-isolating the washed v esicles by centrifugation at 40,000 g for 3 hrs and do not perform any further purification steps.We refer to these as vesicle-enriched secretomes (VESs).It is possible ho w e v er that these pr epar ations hav e other cellular a ggr egates or cellular components co-isolated as well.On the other hand, the soluble part of the bacterial secretome will not be co-isolated using this protocol.

Measuring the effect of VESs on growth of different bacterial species
The effect of the v esicle-enric hed secr etomes on gr owth of different bacteria was measured by analyzing the gr owth curv es obtained for each bacterial species in the absence and presence of VES.These gr owth curv es wer e obtained using a Bioscr eenC analyzer at OD 600 , with measurements taken every 4 min.Overnight gr own cultur es wer e diluted 1 : 1000 in an a ppr opriate medium either with or without the VES. 4 biological r eplicates wer e used in each case .T he exponential growth rate was measured by fitting the OD 600 values between 0.02 and 0.09 to an exponential growth equation N = N * e rt using the KaleidaGr a ph softwar e, wher e r (min −1 ) r epr esents the exponential gr owth r ate .T he stationary phase density was calculated using the R-pac ka ge gr owthcurv er (Sprouffske and Wagner 2016 ).

Measuring bacterial fitness in two-and three-species systems
To perform competition experiments in two-and three-species systems , o v ernight cultur es for eac h str ain wer e mixed in equal ratios (b y v olume), and the competition w as performed in 200 μl of media.In each case, the competition experiments were performed either in the absence or the presence of VESs .T he experiment was done over a period of 2 da ys , with 1 μl of the ov ernight-gr own cultur es tr ansferr ed to 200 μl of the a ppr opriate fr esh media (i.e.either with or without VESs) each day.Different concentrations of VESs were added in the competition experiments to determine how these would affect the competitive ability of the less-fit bacteria in the comm unity, wher e the differ ent concentr ations of VESs r epr esent differ ent v alues of the phenotype of inter est.T hus , for the two-species system, 4X-( ∼ 8.35E + 9 particles/ml) and 8X-( ∼1.67E + 10 particles/ml) concentrated VESs were used; while for the three-species system, 8X-( ∼4.965E + 10 particles/ml) concentrated VESs was used.The association between this and the fitness (the competitive ability) of the bacteria would determine the r ele v ance of this phenotype in micr obial comm unity inter actions.Ov er the course of the experiment, the frequencies of different strains were measured by tagging different bacteria with genes that encoded fluorescent proteins.In the three species system, Esc heric hia coli , Salmonella Typhim urium LT2 and Bacillus subtilis were tagged with y ello w, blue and red fluorescent pr oteins, r espectiv el y, and the c hanges in fr equencies of str ains over the experiment were measured as changes in the signals of the differ ent fluor escence markers .In the two-species systems , Salmonella Typhimurium LT2 was tagged with a y ello w fluorescent protein, while Salmonella arizonae was untagged, and the change in frequency of strains over the experiment was measured as the changes in the fluorescence to the non-fluorescence signal.The genes encoding the y ello w and blue fluorescent proteins were inserted in E. coli and S. Typhim urium, r espectiv el y, using the λ Red system as has been described else wher e (Elowitz et al. 2002 ), while the B. subtilis strain expressing the red-fluorescent pr otein was fr om Ák os T K o vács (Microbiome Ecology at Leiden University, Institute of Biology) (van Gestel et al. 2014 ).The frequencies of each strain were determined every 24-hrs .T his was done by making 100-fold dilutions of cultures from each well in phosphate-buffered saline.100,000 cells were counted in each case and the fraction of differently labelled cells was determined b y flo w c ytometry (MACSQuant VYB, Miltenyi Biotec).Eight biological r eplicates wer e used in eac h case .T he selection coefficient (s) was calculated by plotting natural logs of ratios of population sizes to time, and by calculating the slope of the linearly r egr essed line suc h that s = [ln(N strain1_t_final /N strain2_ t_final )ln(N strain1_t_initial /N strain2_ t_initial )]/(t_final-t_initial) (Dykhuizen 1990 ), where N stands for the population size of the respective strains, while t_initial and t_final are the two time-points where the population sizes were measured.

Experimental evolution in the presence of VES
To determine the ada ptiv e r esponse of micr obial populations tow ar ds the antagonistic effects of different VES, we either used E. coli K-12 MG1655 or S .Typhimurium LT2.The former was serially passaged in the presence of VES isolated from ECOR 11 or ECOR 36, while the latter was serially passaged in the presence of VES isolated from itself.Under each condition, four independent linea ges wer e seriall y passa ged.The e volution experiment was performed in 1 ml culture volume, with 1 μl being tr ansferr ed e v ery day to the fresh media, and with VESs being added to the media e v ery day.Measur ements on gr owth-r ates wer e done e v ery 30 gener ations, and the e volv ed populations wer e stor ed at −80 • C. The experiments were conducted for one-week, during which time the VES pr epar ations wer e stor ed at 4 • C.

Whole-genome sequencing of clones resistant to antagonistic v esicle-enric hed secretomes
Clones that had become resistant to w ar ds the growth inhibition caused by the VES were whole genome sequenced.DNA was extr acted fr om 1 ml ov ernight cultur es using the MasterPur e Complete DNA & RNA Putification Kit (Epicentre) according to the manufacturer' s instructions.Illumina' s Nextera XT kit was used to make libraries (2 ×300) that were sequenced on Illumina's Miseq platform.Samples were dual-indexed and pooled together .Average whole genome coverage per sample was ∼30X.Analysis of the fastq files obtained from Miseq sequencing was performed using CLC genomics Workbench version 8 and were mapped to the reference genome of the ancestral S .Typhimurium LT2 strain or E. coli genome.SNP calling and structur al r earr angements wer e both assessed using this tool.

Cloning of ColE1 immunity gene
To determine the association between colicins and vesicles, the ColE1 immunity protein was cloned in the IPTG-inducible plasmid pCA24N.Plasmids isolated from ECOR 36 were used as a template to amplify the immunity gene.For the PCR, each primer consisted of a 3 -end corresponding to the annealing site, while 5overhangs contained the restriction enzymes sites for KpnI and PstI .Amplified PCR pr oducts wer e purified using the Qiagen purification kit and then ligated using a T4-ligase .T he ligations were then electroporated in E. coli K-12 MG1655 and selected on c hlor amphenicol containing plates.Ten clones were isolated and the presence of the cloned ColE1 gene was confirmed using local Sanger sequencing.

Sta tistical anal ysis
To determine the statistical significance of our data, we either performed an unpaired two-sided Student's t-test or an ANOVA analysis follo w ed b y a Tuk e y's HSD test (post hoc analysis).T hus , the ttest was performed to determine the effect of VESs on the growth of single species of bacteria ( Fig. S1 ), to determine the effect of VESs on three-species competition experiments (Fig. 3 ), to determine the effect of different nutrient types on VES-mediated micr obial inter actions (Fig. 6 ), to determine the association of VESs with different types of colicins ( Fig. S3 ), and to determine the gr owth differ ences in m utants r esistant to the anta gonistic ef-fects of VESs (Figs 4 and 5 ).Wher e v er a ppr opriate, Bonferr oni's corr ection was a pplied to corr ect for m ultiple comparisons .T he adjusted p-value was obtained by dividing the calculated p-value by the number of comparisons performed.
A one-w ay ANOVA, follo w ed b y a Tuk e y HSD post hoc analysis, was performed to determine the effect of VESs on twospecies competition experiments (the different levels of VESs were considered a factor, Fig. 2 ) and to determine the role of genetic bac kgr ound on VES-mediated microbial interactions (the different host bac kgr ound wer e consider ed a factor, Fig. S4 ).
All the data and the analysis outputs (including p-values, tvalues, F-statistic and adjusted P -values) are provided in Tables S3 -S6 .

Effects of VESs on the growth of different species of bacteria
To determine how VESs affect the growth of different species of bacteria in l ysogen y br oth (LB) gr owth media, nine differ ent isolates of bacteria (four isolates of Esc heric hia coli , two isolates of Salmonella enterica , two isolates of Pseudomonas aeruginosa, and one isolate of Bacillus subtilis ) were studied in the presence and absence of VES isolated from each of the bacteria ( Fig. S1 ).Instead of measuring these effects by normalizing the stationary phase bacterial density (to k ee p the n umber of bacterial cells constant acr oss differ ent comparisons) or by normalizing to the number of vesicles produced per bacterial species, these effects wer e measur ed by k ee ping the v olume of gro wth media constant.This was done since both the stationary phase bacterial density and the number of vesicles produced per bacterial species is a function of a giv en envir onment, and both might play a role in determining the effect of VESs on bacterial interactions.T hus , we consider k ee ping the growth environment, volume of growth media, and total growth time constant as the most appropriate setup to determine the relevance of the VES to bacterial interactions.
The concentration of the vesicles used in our assay was measured using the nanoparticle tracking analysis method and r anged fr om ∼ 10 10-10 1 particles/ml (Table S2).In eac h case, we measured how the VESs affected the exponential growth rate and the stationary phase population density of the bacteria.All the values for these growth parameters are presented relative to values obtained for the growth of the bacteria in the absence of the VESs.To be conserv ativ e in our initial scr eening, we consider onl y those effects to be biologically relevant that either affected these gr owth par ameters by at least 10%, or those where the relative change in the growth parameters between the presence and absence of VESs was statistically significant at P < 0.001 (Two-sided Student's t-test).Our results show that the antagonistic effects of VESs were species-specific ( Fig. S1 , and Tables S3 and S4 ).Out of a total of 162 comparisons, the VESs affected the exponential gr owth r ate in 7 cases (5 statisticall y significant at P < 0.001, corrected for multiple testing using Bonferroni's correction, and 2 having an effect of ∼10% or more; the latter includes the effect of VESs of P. aeruginosa PA14 on S. arizonae, and the effect of VESs of B. subtilis on itself; Table S3) and the stationary phase density in 17 cases (12 statistically significant at P < 0.001, corrected for multiple testing using Bonferroni's correction, and 5 having an effect of ∼10% or more; the latter include the effect of VESs of P. aeruginosa PA14 on E. coli K-12 MG1655, the effect of VESs of B. subtilis on S .Typhimurium LT2, the effect of VESs of S .Ty-   phimurium LT2 on S .Typhimurium LT2, the effect of VESs of S. arizonae on S .Typhimurium LT2, and effect of VESs of P. aeruginosa PA14 on S .Typhimurium LT2.Data is provided in Tables S4 ).
The VES isolated from P. aeruginosa PA14 had antagonistic effects on the growth of the highest number of bacterial species tested (se v en in total, 3 where exponential growth rate was affected and 4 where stationary phase density was affected, Fig. S1H ), while those isolated from natural isolates of E. coli i.e.ECOR 11 and ECOR 36 had a strong antagonistic effect on the growth of E. coli K-12 MG1655 ( Fig. S1C and D ).The VESs from S. arizonae and B. subtilis both displayed antagonistic effects on growth of S. Typhimurium LT2 ( Fig. S1F and I ).Inter estingl y, S. Typhim urium LT2, E. coli ECOR 36, and S. arizonae all displayed self-inhibition due to their VESs ( Fig. S1 ).Typhim urium LT2 (triangles) measur ed under differ ent growth conditions in the presence of VESs isolated from ECOR 36, S. arizonae and P. aeruginosa PA14.The different growth conditions used were either nutrient-ric h l ysogen y br oth (LB) or nutrient-poor glucose (0.2%) M9-minimal media (Glu MM).All gr owth par ameters ar e shown normalized to v alues obtained for the ancestral strain grown in the absence of the VES.A two-sided Student's-t test was performed to determine statistical significance.* indicates statistical significance at P < 0.001 when growth parameters are compared to parameters of ancestral strain grown in the absence of VESs, while * indicates statistical significance when the normalized parameters are compared across conditions.Error bars represent the standard deviation in each case .T he concentration of the VESs is mentioned in Table S2.

Fitness-phenotype maps show changes in the competiti v e a bility of bacterial species with changes in levels of VES
We wanted to determine if the VES that displayed antagonistic effects in the single-species experiments would also have similar effects on fitness when the bacterial species were present in multispecies systems.To this end, we generated fitness-phenotype maps for different species of bacteria in two-and three-bacterial species systems, both in the presence and absence of VESs.The two-species system studied was that of S. Typhimurium LT2 and S. arizonae , and the three-species system was that of B. subtilis, E. coli K-12 MG1655 and S. Typhimurium LT2.These systems were chosen for two reasons; first, in our single-species experiments ( Fig. S1 ), the VESs from S. arizonae were antagonistic to w ar ds S. Typhimurium LT2, while those isolated from B. subtilis were antagonistic to w ar ds S. Typhim urium LT2.Secondl y, in eac h case VESs were added from the species that was the least fit in two-and three-species competition experiments , i.e . in the two-species system S. arizonae was outcompeted by S .Typhimurium LT2, while in the three-species system B. subtilis was outcompeted by E. coli and S .Typhimurium LT2.
In the two-species system, S. Typhimurium LT2 outcompeted S. arizonae with a selection coefficient of 0.062 ± 0.008 (Fig. 2 , Tables S5 ).Since S. arizonae was the less dominant of the two species, we next investigated the outcome of competition between the same two species in the presence of VES isolated from S. arizonae .Her e, the pr esence of the VES incr eased the competitiv e adv anta ge for S. arizonae (Fig. 2 ).T hus , incr easing the concentr ation of the VES isolated from S. arizonae by 4-fold resulted in the selection coefficient reducing from 0.062 ± 0.008 to 0.041 ± 0.003 and by 8-fold resulted in a reduction of the selection coefficient to 0.027 ± 0.008 (statistically significant for one-way ANOVA F = 45.88,P = 2.17E-08).Statistical significance between the three conditions was further determined by performing a Tuk e y's HSD (indi vidual P -v alues pr ovided in Table S5).In the thr ee-species bacterial sys-tem ( B. subtilis, E. coli K-12 MG1655 and S. Typhimurium LT2), B. subtilis was the least dominant bacteria under the conditions tested.T hus , when starting with an initial ratio of 1 : 1 : 1, B. subtilis was outcompeted with a selection coefficient of −3.6 ± 0.72 (Fig. 3 ).Ho w e v er, incr easing the concentration of the VES isolated from B. subtilis by 8-fold resulted in a drop of the selection coefficient to −0.77 ± 0.46 (t 6.65,14 , P = 1.93E-07,Table S5 ), resulting in its maintenance in the population.These results show that bacterial VESs have the potential to alter the competitive abilities of bacterial species in microbial communities.

Adapti v e responses towards antagonistic effects of VESs
The single-and multi-species experiments demonstrated that the antagonistic effects of VESs can increase the competitive fitness of v esicle-pr oducing bacterial species .T his observ ation r aises the k e y question of if and how the neighboring bacteria can e volv e in response to the antagonistic VESs.To this end, we employed an experimental e volution a ppr oac h and examined the e volutionary responses to the VESs.We chose the VESs isolated from bacterial species ECOR 11 and ECOR 36 on E. coli K-12 MG1655 because they had the largest effect on the growth of other bacterial species ( Fig. S1C and D ).In each case, the ancestral E. coli K-12 MG1655 str ain was seriall y passa ged in a gr owth envir onment containing the VES from either ECOR 11 or ECOR 36.Evolved clones that were isolated after ∼30 generations of growth displayed resistance tow ar d the antagonistic effects of the VES.The clones that were resistant to the vesicle-enriched secretome isolated from ECOR 11 had a complete r ecov ery of the exponential growth rate (as compared to the ancestral E. coli K-12 MG1655 in the presence of the VESs, Fig. 4 B and Table S6 ), while clones resistant to vesicleenric hed secr etome isolated fr om ECOR 36 onl y had partial r ecovery of the exponential gr owth r ate (as compar ed to the ancestral E. coli K-12 MG1655 in the presence of the VESs, Fig. 4 B and Table S6 ).The VES isolated from ECOR 11 did not have a strong effect on the stationary phase density of the ancestral E. coli K-12 MG1655 ( Fig. S1C ), and we did not observe any difference between the stationary phase density of the resistant clones as compared to the ancestr al str ain in the presence of the VES (Fig. 4 B and Table S6 ).On the other hand, VES isolated from ECOR 36 had a strong effect on the stationary phase density of the ancestral strain (relative stationary phase density = 0.43 ± 0.3, Table S6), and the stationary phase density observed for the resistant mutants in the presence of the VES had r ecov er ed to v alues corr esponding to that of the ancestral E. coli K-12 MG1655 in the absence of the VES (Fig. 4 B and Table S6 ).
To determine the underlying genetic basis for this adaptive response, thr ee r esistant clones wer e whole genome sequenced.Single m utations wer e observ ed in two of these clones that in each case resulted in loss of function of the btuB gene, while the third clone had a deletion of a ∼4.5 kb region that included the tolC gene (Table 1 , Fig. 4 C).The btuB gene encodes a cobalamin outer membr ane tr ansporter pr otein, while tolC encodes an outer membr ane c hannel pr otein that forms a part of the common efflux system of the cell.Of r ele v ance her e, both these pr oteins ar e also the entry site for colicins that are toxins secreted by different natural isolates of E. coli .Both ECOR 11 and ECOR 36 are known colicin producers (Riley and Gordon 1992 ), carrying the ColE2 and the ColE1 type plasmid, r espectiv el y.This suggests that the antagonistic effects of the VES from these natural E. coli isolates might be due to the association between the vesicles and colicins.To test this hypothesis, the colE1 immunity gene that encodes the imm unity pr otein a gainst the colicin ColE1 was cloned on an IPTGinducible plasmid and the plasmid was then transformed into the susceptible ancestral E. coli K-12 MG1655.When the ancestral strain carried the immunity protein it was more resistant to the antagonistic effects of the VES isolated from ECOR 36, as compared to when it carried an empty vector ( Fig. S2 ), confirming that the antagonistic effects of the VESs from the natural isolates of E. coli were due to the associations between colicins and the vesicles.

Different types of colicins are enriched with membrane vesicles in a host-specific manner
To determine whether or not membrane vesicles are enriched with different types of colicins, we isolated the VESs from other colicin-producing E. coli strains in the ECOR strain collection.These included ECOR 12 (ColE1), ECOR 14 (Col1a), ECOR 25 (ColB), ECOR 38 (Col1a), ECOR 41 (ColK and/or N), ECOR 42 (ColB), ECOR 48 (Col1b), ECOR 60 (ColE2) and ECOR 62 (ColB).Vesicle-enriched secr etomes isolated fr om all the colicin pr oducers affected gr owth of E. coli K-12 MG1655 ( Fig. S3 ), although to differ ent le v els .T he strongest effect was observed for the VES isolated from ECOR 41 carrying the colicins ColK and/or N and from ECOR 60 carrying colicins ColE2 (no growth observed in either case), while those isolated from ECOR 25 (ColB) and ECOR 38 (Col1a) had the least effect (r elativ e exponential gr owth r ate of 0.93 ± 0.02 and 0.92 ± 0.04, r espectiv el y, and r elativ e stationary phase density of 0.99 ± 0.008 and 0.99 ± 0.005, r espectiv el y).Inter estingl y, e v en when the ECOR isolates wer e secr eting the same type of colicin, the anta gonistic effect of their VES varied.T hus , the VESs of ECOR 36 and ECOR 12 (both expressing the Col E1) and of ECOR 25, ECOR 42, and ECOR 62 (all expressing the ColB) displayed different antagonistic effects ( Fig. S4 ).Among colicin B pr oducing str ains, ECOR 42 had the str ongest anta gonistic effect.The effect of VESs isolated from these strains on relative stationary phase density was statisticall y differ ent fr om one another, while onl y ECOR 42 had a statis-tically significant effect on the relative exponential growth rate of E. coli K-12 MG1655 ( Fig. S4 ; F-v alues, P -v alues for one-way ANOVA and adjusted p-values for Tuk e y's HSD gi ven in Tables S3 and S4 ).Among colicin E1 pr oducing str ains, VESs isolated from ECOR 36 had a m uc h str onger and statisticall y differ ent effect as compar ed to ECOR 12 ( Fig. S4 ; F-v alues, P -v alues for one-way ANOVA and adjusted P -values for Tuk e y's HSD gi ven in Tables S3 and S4 ).These results suggest that the antagonistic effects of the VESs, besides being dependent on the causal protein itself, also depend on the genotype of the bacteria.

Evolution in response towards self-inhibition from v esicle-enric hed secretomes
In the initial analysis of the effect of VESs on the growth of single species of bacteria, se v er al cases of self-inhibition wer e observ ed.To determine the underlying mechanism for self-inhibition, we inv estigated the e volutionary r esponses of S .Typhim urium LT2 when it was exposed to its own VES.Evolved clones isolated fr om populations passa ged in the pr esence of the VESs for ∼70 gener ations of gr owth demonstr ated r esistance to w ar ds the selfinhibition (Fig. 5 A).All three mutants that were resistant to selfinhibition by the v esicle-enric hed secr etome had a higher relative stationary phase density (statistically different at P < 0.001 for a two-sided Student's t-test; Table S6), while only one of the clones had a higher r elativ e exponential gr owth r ate (statisticall y differ ent at P < 0.001 for a two-sided Student's t-test; Table S6 ).
To determine the mechanistic basis for this observed reduced susceptibility, we whole genome sequenced three resistant clones (Table 2 ).In two of the resistant clones, large-scale genome amplifications spanning a 100-kb region in one resistant clone, and a 200-kb region in the other resistant clone were observed.Although these amplifications were observed in overlapping regions of the genome (Fig. 5 B, Table 2 ), given the large number of genes involved within these amplifications the genetic basis of resistance is still unclear in these clones .In the third resistant clone, we observed a non-synonymous mutation in the gene ftsW (Leu186Pro).Since this was the onl y m utation found in this clone, it dir ectl y links the protein FtsW in conferring resistance to self-inhibition caused by the VES.The ftsW gene codes for an essential cell-division protein that functions as a peptidogl ycan gl ycosyltr ansfer ase .T his suggests that the self-inhibitory effect of the VESs observed in S. Typhimurium LT2 could be a result of the cargo in the membrane v esicles inter acting with pr oteins involv ed with bacterial cell division.

VES-mediated microbial interactions are dependent upon the type of nutrients in the environment
To determine how microbial interactions caused by the VES vary with a change in the type of nutrients in the environment, VESs of bacteria grown in nutrient-poor glucose minimal media were investigated for their effect on the growth of other bacterial species .T hus , VESs were isolated from E. coli ECOR 36, S. arizonae and P. aeruginosa PA14 after growth in M9-glucose minimal medium, and their effect was studied on growth of E. coli K-12 MG1655 and S. Typhimurium LT2 .These sets of bacterial combinations wer e specificall y c hosen since under nutrient-rich conditions (i.e. in LB), VESs from E. coli ECOR 36 and P. aeruginosa PA14 had the strongest effect on E. coli K-12 MG1655 ( Fig. S1D and H ), while v esicle-enric hed secr etome fr om S. arizonae and P. aeruginosa  (PA14) displayed a strong effect on S. Typhimurium LT2 ( Fig. S1F  and H ) .
Ov er all, the gr owth medium had a str ong effect on VESmediated micr obial inter actions (Fig. 6 ).VESs isolated fr om ECOR 36 had no effect on either the exponential growth rate or the stationary phase density of E. coli K-12 MG1655 in the nutrient-poor glucose minimal medium, as compared to the nutrient-rich LB medium where it had strong effects on both these growth par ameters.Similarl y, the VESs of S. arizonae grown on nutrient-poor medium had no effect on the growth of S .Typhimurium LT2, instead of the antagonistic effect that was observed under nutrientrich conditions (Fig. 6 and Tables S3 and S4 , r elativ e stationary phase density statistically different at P < 0.001 for a two-sided Student's t-test).On the other hand, vesicles from P. aeruginosa had a m uc h str onger anta gonistic effect on both E. coli K-12 MG1655 and S. Typhimurium LT2 in nutrient-poor medium as compared to nutrient-rich LB medium (Fig. 6 and Tables S3 and S4 , statistically different at P < 0.001 for a two-sided Student's t-test), such that the bacteria displayed no growth at all in the presence of the VES in the former medium.Although our r esults conclusiv el y show that VES-mediated microbial interactions are dependent on the type of nutrient in the envir onment, mor e work is needed to understand whether this is due to a change in the properties of the vesicles, or due to a change in physiology of the bacteria itself, or both.

Discussion
Membr ane v esicles and v esicle-enric hed secr etomes hav e mainl y been studied due to their potential medical significance, either as carriers of virulence factors (Kesty et al. 2004, Kaparakis-Liaskos and Ferr er o 2015, Koeppen et al. 2016, Nice et al. 2018 ) or as vaccine deliv ery v ehicles (v an der Pol et al. 2015, Cheng et al. 2021 ), and it is only recently that their ecological relevance and importance in bacterial communities have been examined.Our results show that VESs can indeed have strong effects on microbial community composition by altering the competitive abilities of the bacteria present.T hus , in the two-and three-species systems examined here, the addition of VESs from the bacterial species that w as a w eak competitor resulted in its maintenance in the comm unity, whic h was in part due to the antagonistic effects of VESs to w ar d other bacterial species .Furthermore , using an experimental evolution approach, we showed that these VESs may act as selectiv e pr essur es that influence the composition of the microbial community.

Significance of membrane VESs as antagonistic agents in microbe-microbe interactions
VESs carry different types of proteins that can influence the growth of both the producing and neighboring bacterial species.We observ ed se v er al cases of anta gonistic effects of VESs, and in one case demonstrated that the observed antagonism was due to colicins, a group of ecologically relevant toxins .T hus , the vesicleenric hed secr etomes of all the natur al isolates of E. coli that contained a colicin-producing plasmid displayed antagonism against E. coli K-12 MG1655.Colicins ar e gener all y secr eted as soluble pr oteins in the bacterium's environment; thus in their soluble state, they would not be co-isolated with the membrane vesicles using the method we have used to enrich vesicles .T hus , this observation suggests a novel association between colicins and membrane v esicles.Inter estingl y, the anta gonistic effect observ ed fr om these VES was dependent on the genotype of the colicin-producing bacteria, such that the bacteria secreting the same type of colicin exhibited different levels of growth inhibition.Although this could either be due to different levels of colicin being expressed, different amounts of vesicles being secreted, or different levels of association between vesicles and colicins among different isolates, this result highlights the important role of the bacterial genotype in effecting the properties of VESs.
Anta gonistic effects wer e also observ ed for the VESs isolated from P. aeruginosa PA14.Membrane vesicles from P. aeruginosa have been extensiv el y studied, and ar e known to carry compounds that can affect both eukaryotic and prokaryotic cells .T hese studies have shown that microbial interactions mediated by the vesicles isolated from P. aeruginosa are largely observed due to the association of antibacterial quinolones , iron-sca venging proteins , autolysin enzymes, and quorum-sensing inducers with the vesicles (Kadurugamuwa and Beveridge 1996, Tashiro et al. 2012, Tashiro et al. 2013, Cooke et al. 2019 ).Our results corroborate the observ ations fr om earlier studies in demonstr ating the anta gonistic effects of membrane vesicles from P. aeruginosa on a variety of bacterial species.

Environments containing different nutrient alter VES-mediated microbial interactions
For a given species of bacteria, the number of vesicles produced and the cargo present within these vesicles can vary with growth conditions (Macdonald andKuehn 2013 , Orench-Rivera andKuehn 2016 ).For example, membrane vesicles isolated from Pseudomonas putida KT2440 grown in benzoate-containing minimal media contained benzoate-degrading enzymes whic h wer e absent in membrane vesicles when the bacteria w ere gro wn in nutrient-ric h l ysogen y br oth (Choi et al. 2014 ).Se v er al studies have also shown increased vesicle production under stressful conditions, which is either a defense mec hanism a gainst these stresses or an indirect effect of other stress response pathwa ys .Important roles of membrane vesicles have been suggested in maintaining homeostasis under conditions leading to membrane str ess, pr otein misfolding, and nutrient starvation (Nagakubo et al. 2019 ).Besides this, the presence of antibacterial agents, compounds that affect the pH of the system, the presence of phages, and non-optimal temper atur es may increase vesiculation in bacteria (Manning andKuehn 2011 , de Jonge et al. 2021 ), implying that microbial interactions that are mediated through vesicleenric hed secr etomes also depend on the envir onment.Results from this study support this assertion, where two instances of VESs-mediated interactions changed with alterations in the environmental conditions .T hus , the VES isolated from P. aeruginosa PA14 sho w ed differ ent le v els of anta gonism to w ar ds E. coli K-12 MG1655 and S. Typhimurium LT2 based on nutrient le v els, whereas the effects of VES isolated from S. arizonae on growth of S. Typhimurium went from being antagonistic under nutrient-rich conditions to having no effect under nutrient-poor conditions.These results highlight how the type of nutrient in an environment can influence the effect of the VESs on microbial community dynamics.

VESs can result in self-inhibition of the producing species
We also observed self-inhibitory effects of the v esicle-enric hed secr etomes, wher e the v esicle-enric hed secr etomes isolated fr om S. Typhimurium, E. coli ECOR 36, and S. arizonae resulted in selfinhibition.Although the ecological or evolutionary relevance of self-inhibition by vesicles is unclear, our work demonstrates that mutants that are resistant to such self-inhibitory effects could r a pidl y be selected since resistant S .Typhimurium LT2 mutants emer ged in pr esence of its own v esicle-enric hed secr etome.One of these resistant mutants had a mutation in a cell-division protein FtsW, suggesting that the self-inhibiting effects might be due to the interaction of the v esicle-enric hed secr etome with the celldivision machinery.

Conclusion
Membr ane v esicle pr oduction is a common phenotype observ ed in all bacteria and by tying bacterial fitness to membr ane v esicle production, our study has taken an initial step in elucidating the mechanisms by which vesicle-enriched secretomes can affect community dynamics.To demonstrate these effects, we have used vesicle-enriched secretomes where the vesicles were concentrated ∼ 8-fold.It is notable that se v er al m utations hav e been shown to individuall y incr ease the le v el of v esicle pr oduction (Kulp et al. 2015 ).Consequently, an important next step to understand the r ele v ance of these vesicles in microbial communities would be to identify ecological conditions wher e m utations that c hange c har acteristics (amount, composition) of the v esicleenric hed secr etome ar e enric hed by selection.Suc h studies will further increase our understanding of the evolutionary and ecological significance of these vesicles.

Figure 1 .
Figure 1.Determining the role of vesicle-enriched secretomes (VESs) on bacterial interactions.(A) Schematic presentation of the preparation of v esicle-enric hed secr etomes fr om differ ent bacterial species .T he pr epar ations contain membr ane v esicles that carry differ ent types of cellular biomolecules.(B) VESs can affect the dynamics of a microbial community by mediating antagonistic and synergistic microbial interactions.

Figure 2 .
Figure 2. Effect of v esicle-enric hed secr etome (VES) isolated fr om S. arizonae on competition between S .Typhimurium LT2 and S. arizonae .Three differ ent concentr ations of v esicle-enric hed secr etomes wer e used to determine their effect on the competitive ability of S. arizonae .The concentration of the 8-fold concentrated VESs used was 1.67E + 10 particles/ml.Y-axis shows the selection coefficient measured during competition between S.Typhimurium LT2 and S. arizonae ( S. Typhimurium LT2/ S. arizonae ) in l ysogen y br oth at 37 • C, while the X-axis shows differ ent concentr ations of VES used.Eight replicates were used for each concentration.A one-way ANOVA analysis was performed to determine statistical significance for the effect of VESs on selection coefficients (F = 45.88,p = 2.17E-08).This was followed by performing a Tuk e y's HSD test to determine significant differences between different VES concentrations, where * indicates statistical significance at P < 0.01.The median of the data set is represented as a horizontal line.

Figure 3 .
Figure 3.Effect of v esicle-enric hed secr etome (VES) isolated fr om B .subtilis on competition between B .subtilis, E. coli K-12 MG1655 and S .Typhimurium LT2.Competition experiments were performed in 3-species systems consisting of B. subtilis , E. coli K-12 MG1655 and S. Typhimurium LT2 in lysogeny broth at 37 • C, in the absence or presence of VES isolated from B. subtilis .Y-axis shows the selection coefficient measured during competition between these three strains ( B. subtilis / E. coli + S .Typhimurium) while X-axis shows different concentrations of VES used.Eight replicates were used for each concentr ation.An unpair ed two-sided Student's-t test was performed to determine statistical significance and * indicates statistical significance at P < 0.001.The median of the data set is r epr esented as a horizontal line.The concentration of the VESs used was 4.965E + 10 particles/ml (8-fold concentrated).

F igure 4 .
Ev olution of E. coli K-12 MG1655 populations growing in the presence of antagonistic vesicle-enriched secretomes (VESs).(A) Experimental evolution design for selection of mutants resistant to the antagonistic effects of VESs .T he evolution experiment is carried out in lysogeny broth at 37 • C, and has VES added to the media at each cycle.(B) Relative exponential growth rate and relative stationary phase density are shown for E. coli m utants r esistant to anta gonistic effects of VES isolated fr om ECOR 11 and ECOR 36.All gr owth par ameters ar e shown normalized to v alues obtained for the susceptible ancestral strain grown in the absence of the VES.Four replicates are used in each case.A two-sided Student's-t test was performed to determine statistical significance and * indicates statistical significance at P < 0.001 when comparing the parameters for the ancestral strain to e volv ed r esistant m utants in the pr esence of v esicle-enric hed secr etomes.Err or bars r epr esent the standard de viation in eac h case .T he concentration of the VESs used in these experiments is 4.16E + 10 for VESs isolated from ECOR 11 and 8.375E + 10 for VESs isolated from ECOR 36 (Table S2).All experiments were performed in lysogeny broth at 37 • C. (C) Mutations were identified in E. coli mutants resistant to the antagonistic vesicle-enriched secr etomes.Deletions ar e shown in r ed, while point m utations ar e shown in blue.F igure 5. Ev olution of S. Typhimurium LT2 to w ar ds self-inhibition due to antagonistic vesicle-enriched secretomes (VESs).(A) Relative exponential gr owth r ate and r elativ e stationary phase density ar e shown for S. Typhim urium LT2 m utants r esistant to self-inhibition by anta gonistic VES isolated fr om S. Typhim urium LT2.All gr owth par ameters ar e shown normalized to v alues obtained for the susceptible ancestr al str ain gr own in the absence of the VES.For the measurement of relative exponential growth rates and relative stationary phase density, two replicates are used.A two-sided Student's-t test was performed to determine statistical significance and * indicates statistical significance at P < 0.001 when comparing the parameters for the ancestral strain to evolved resistant mutants in the presence of vesicle-enriched secretomes.Error bars represent the standard deviation in each case .T he concentration of the VESs used is 5.885E + 10 particles/ml (Table S2).All experiments were performed in lysogeny broth at 37 • C. (B) Mutations identified in S. Typhimurium LT2 mutants resistant to the antagonistic vesicle-enriched secretomes.Amplifications are shown in green, and point mutations are shown in blue.

Figure 6 .
Figure 6.Dependence of antagonistic effects of vesicle-enriched secretomes (VESs) on the nutrients in the environment.(A) Relative exponential gr owth r ate and (B) r elativ e stationary phase density for E. coli K-12 MG1655 (circles) and S. Typhim urium LT2 (triangles) measur ed under differ ent growth conditions in the presence of VESs isolated from ECOR 36, S. arizonae and P. aeruginosa PA14.The different growth conditions used were either nutrient-ric h l ysogen y br oth (LB) or nutrient-poor glucose (0.2%) M9-minimal media (Glu MM).All gr owth par ameters ar e shown normalized to v alues obtained for the ancestral strain grown in the absence of the VES.A two-sided Student's-t test was performed to determine statistical significance.* indicates statistical significance at P < 0.001 when growth parameters are compared to parameters of ancestral strain grown in the absence of VESs, while * indicates statistical significance when the normalized parameters are compared across conditions.Error bars represent the standard deviation in each case .T he concentration of the VESs is mentioned in TableS2.

Table 1 .
Mutations identified in E. coli K-12 MG1655 mutants resistant to antagonistic effects of membrane vesicle-enriched secretomes isolated from ECOR 11 and ECOR 36.
* Indicates a stop codon.

Table 2 .
Mutations identified in S. Typhimurium LT2 mutants resistant to self-inhibitory effects of membrane vesicle-enriched secretomes.